Project Summary/Abstract The combination of doxorubicin (adriamycin) and cyclophosphamide, so-called AC chemotherapy, is commonly used for the clinical treatment of breast and other cancers. DNA apurinic/apyrimidinic (AP) sites are a common result of alkylating agents such as nitrogen mustards. The cytotoxicity of alkylating agents can be enhanced by methoxyamine, which forms a conjugate with AP sites and inhibits repair. We propose a new reactivity of doxorubicin and related agents in which they form a covalent conjugate with AP sites in DNA, and this adduct is highly cytotoxic. The following interconnected Aims are proposed to address this hypothesis. Project 1 will demonstrate that the anthracycline antitumor agents doxorubicin, mitoxantrone and pixantrone form covalent conjugates with AP sites in duplex DNA through an imine linkage (Specific Aim 1). Imines linkages form reversibily; we therefore propose to synthesize new analogues that form more stable hydrazine, semicarbazone, and oxime covalent linkages to AP sites. Nitrogen mustards cause complex forms of DNA damage. In order to evaluate the biological processing of nitrogen mustard DNA adduct, we will synthesize structurally defined oligonucleotides that contain DNA adducts of nor-nitrogen mustard (the active form of cyclophosphamide) and thioTEPA. This includes N5-substituted formamidopyrimidine-dG adducts derived from imidazole ring-opened of the cationic N7-alkylated dG (Specific Aim 2). We will also synthesize complex secondary products from nitrogen mustards cross-links in which one of the cross-linked dGs has undergone deglycosylation to an AP site. Based on deglycosylation rates, we predict thioTEPA and nor- nitrogen mustard will lead to a high burden of AP sites compared to other alkylating agents. The extent to which these alkylating agents form AP sites in DNA in vitro (calf thymus DNA and cultured human breast cancer cells) and in vivo (rodent models and white blood cells from breast cancer patients undergoing AC chemotherapy) as well as AP site conjugates of doxorubicin and related agents will be quantified by ion trap multistage mass spectrometry (Specific Aim 3). Project 1 will work closely with the DNA Synthesis Resource Core to provide site-specifically modified oligonucleotides containing nor-nitrogen mustard and thioTEPA adducts (Aim 1) as well as covalent drug conjugates of an AP site (Aim 2) to Project 2 for replication, mutagenesis and repair studies, and Project 3 for structural analysis. Project 2 will examine the cytotoxicity of the anthracyclines and new analogues in combination with nor-nitrogen mustard or thioTEPA in cultured human breast cancer cell lines.